Hemoregulatory peptides

ABSTRACT

PCT No. PCT/US92/10070 Sec. 371 Date May 25, 1994 Sec. 102(e) Date May 25, 1994 PCT Filed Nov. 24, 1992 PCT Pub. No. WO93/10807 PCT Pub. Date Jun. 10, 1993The present invention relates to novel peptides which have hemoregulatory activities and can be used to inhibit the myelopoietic system of humans and animals.

This application is a 371 of PCT/US 92/10070 filed Nov. 24, 1992. Thisapplication is also a continuation-in-part of Ser. No. 07/799466 filedNov. 26, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates to novel peptides which havehemoregulatory activities and can be used to inhibit the myelopoieticsystem of humans and animals.

BACKGROUND OF THE INVENTION

A variety of regulatory messengers and modifiers such as colonystimulating factors, interferons, and different types of peptides areresponsible for the regulation of myelopoiesis. Metcalf, Cell, 43:5(1985); Baserga R., Foa P., Metcalf D, Polli EE (eds), BiologicalRegulation of Cell Proliferation (1986); Nicola et al., J. Cell Physiol.128:501 (1986), Zoumbos et al., Proyr. Hemat. 1:341 and 14:201 (1986);Werner et al., Experientia 42:521 (1986). Over twenty years ago, Rytomaaand Kivieniemi Cell Tissue Kinet 1:329-340 (1968); Rytomaa et al.,Control of Cellular Growth in Adult Organisms pp 106-138 (1967) reportedthat extracts of mature granulocytes (granulocytic chalone) couldspecifically inhibit rat myelopoietic cell proliferation in cover slipcultures. Later, they demonstrated that the factor, which had amolecular weight less than 3,000 daltons, was able to induce theregression of a transplantable rat granulocytic leukemia, as well asretard the growth of leukemia cells in humans. Paukovits and othersextracted a similar factor from rat bone marrow cells and showed that itinhibited the titrated thymidine uptake of bone marrow cells, Paukovits,W. R., Cell Tissue Kinet 4:539-547 (1971); Naturforsch 37:1297 (1982).In 1979, Boll et al., Acta Haematologica 6:130 (1979) demonstrated theinhibitory effects of rat granulocytic extracts on human bone marrowcells in culture and a number of other researchers demonstrated thatthis crude granulocytic extract inhibited the development of g-CFUCand/or gm-CFUC in vitro from rodent bone marrow cells.

This biological agent was termed a granulocyte chalone which, accordingto this theoretical concept, should be an endogenous inhibitor of cellproliferation acting in the same tissue as it was secreted. The materialobtained from crude extracts was found to be non-species specific buthighly tissue specific. Furthermore, it was found to be nontoxic and tohave reversible activities.

In 1982, a synthetic hemoregulatory pentapeptide was reported to have aselective inhibitory effect on myelopoietic cells both in vitro and invivo, where the main effect seems to be on myelopoietic stem cells(CFU-gm), Paukovits et al., Z. Naturforsch 37:1297 (1982) and U.S. Pat.No. 4,499,081. This peptide is believed to be an analogue of a naturallyoccurring granulopoiesis inhibition factor which has been found inminute quantities in bone marrow extracts.

We have now found certain synthetic peptides which have a selectiveinhibitory effect on myelopoietic cells in vitro. These peptides, byinhibiting haematopoiesis, and, in particular, granulopoiesis tend toprevent quiescent cells from entering into cell division and so becomingsusceptible to attack by cytotoxic anti-cancer drugs. In addition toproviding a protective function in therapy using cytotoxic drugs, thepeptides may also be used to arrest proliferation of cancer cellsrelated to the myelopoietic system, i.e. myeloid leukemia.

SUMMARY OF THE INVENTION

This invention comprises peptides, hereinafter represented as formula(I), which have hemoregulatory activities and can be used to inhibithaematopoiesis.

The peptides are useful in providing a protective function in therapyusing irradiation and/or cytotoxic drugs, and may also be used to arrestproliferation of cancer cells related to the myelopoietic system, forexample, in the treatment of myeloid leukemia. The peptides may also beused in many clinical situations where it is desirable to alterhemopoiesis.

These compounds may also be used in combination with the dimers ofco-pending U.S. application Ser. No. 071,547,730, incorporated byreference herein, to provide alternating peaks of high and low activityin the bone marrow cells, thus augmenting the natural circadian rhythmof haematopoiesis. In this way, cytostatic therapy can be given atperiods of low bone marrow activity, thus reducing the risk of bonemarrow damage, while regeneration will be promoted by the succeedingpeak of activity. This invention is also a pharmaceutical composition,which comprises a compound of formula (I) and a pharmaceuticallyacceptable carrier.

This invention further constitutes a method for inhibiting themyelopoietic system of an animal, including humans, which comprisesadministering to an animal in need thereof, an effective amount of acompound of formula (I).

DETAILED DESCRIPTION OF THE INVENTION

The peptides of this invention are illustrated by the formula (I):

    A-B-C-D-(X).sub.n -E                                       (I)

wherein:

A is picolinic acid, pyroglutamic acid, proline or L-pipecolinic acid,

B is serine, glutamic acid, aspartic acid, threonine, cysteine ortyrosine;

C is aspartic acid or glutamic acid;

D is aminobutyric acid, alanine, propargyl glycine, glycine, serine,cysteine or β alanine;

X is tyrosine or lysine;

n is 0 or 1;

E is --N(R₁)--R₂ R₃ or E is glycine, lysine, ornithine, tyrosine,para-aminophenylalanine or the carboxamide or hydroxymethyl derivativethereof;

R₁, R₄ and R₅ are independently hydrogen, C₁₋₅ alkyl, phenyl, napthyl orC₅₋₇ cycloalkyl;

R₂ is C₁₋₆ alkyl, phenyl, naphthyl or C₅₋₇ cycloalkyl;

R₃ is --NR₄ R₅ or aziridinyl, pyrrolidinyl, imidazolyl, pyrrolyl,piperidinyl, piperazinyl, morphollinyl, pyrrolidinyl or pyrralinyl;provided the compound of formula (I) is not p-Glu-Glu-Asp-Cys-Lys; or apharmaceutically acceptable salt thereof.

Also included in this invention are pharmaceutically acceptable saltcomplexes of the compounds of this invention. It should be noted informula (I) E comprises the terminal carboxyl group of the amino acidresidue corresponding to glycine, lysine, ornithinepara-aminophenylalanine or the carboxamide or hydroxy methyl derivativesthereof or E is the amide group of the --N(R₁)--R₂ R₃ group.

The abbreviations and symbols commonly used in the art are used hereinto describe the peptides:

ala=alanine

pGlu=pyroglutamic acid

Pro=proline

Glu=glutamic acid

Asp=aspartic acid

Tyr=tyrosine

Pic=picolinic acid

Ppc=L-pipecolinic acid

Abu=aminobutyric acid

Ppg=propargyl glycine

Gly=glycine

Orn=ornithine

p-(NH₂)Phe=para-aminophenylalanine

Hna=2,6-diamino-4-hexynoic acid

Lys=lysine

Cad=cadaverine

In accordance with conventional representation, the amino terminus is onthe left and the carboxy terminus is on the right. All chiral aminoacids may be in the D or L absolute configuration.

The amino terminus may be protected by acylation. Examples of suchprotecting groups are, t-butoxycarbonyl (t-Boc), CH₃ CO and Ar--CO(Ar=benzyl, or phenyl).

The C-terminus may be carboxy as in the case of the natural amino acidor the carboxamide ##STR1## or hydroxymethyl (--CH₂ --OH).

Preferred compounds are those in which:

A is pyroglutamic acid or picolinic acid;

B is glutamic acid or serine;

C is aspartic acid;

D is aminobutyric acid;

E is glycine or lysine; and

n is 0;

and the chiral amino acids are in the L absolute configuration.

Especially preferred is pGlu-Glu-Asp-Abu-Lys (SEQ ID NO: 1) andPic-Ser-Asp-Abu-Lys (SEQ ID NO: 3). The peptides of the invention areprepared by the solid phase technique of Merrifield, J. Am. Chem. Soc.,85, 2149 (1964), or solution methods known to the art may besuccessfully employed. The methods of peptide synthesis generally setforth in J. M. Stewart and J. D. Young, "Solid Phase Peptide Synthesis",Pierce Chemical Company, Rockford, Ill. (1984) or M. Bodansky, Y. A.Klauser and M. A. Ondetti, "Peptide Synthesis", John Wiley & Sons, Inc.,New York, N.Y. (1976) may be used to produce the peptides of thisinvention and are incorporated herein by reference.

Each amino acid or peptide is suitably protected as known in the peptideart. For example, the a-fluoroenylmethyloxycarbonyl group (Fmoc) ort-butoxycarbonyl (t-Boc) group are preferred for protection of the aminogroup, especially at the α-position. A suitably substituted carbobenzoxygroup may be used for the ε-amino group of lysine and benzyl group forthe β and γ carboxy groups of Asp and Glu respectively. Suitablesubstitution of the carbobenzoxy protecting group is ortho and/or parasubstitution with chloro, bromo, nitro or methyl, and is used to modifythe reactivity of the protective group. Except for the t-Boc group, theprotective groups are, most conveniently, those which are not removed bymild acid treatment. These protective groups are removed by such methodsas catalytic hydrogenation, sodium in liquid ammonia or HF treatment asknown in the art.

If solid phase synthetic methods are used, the peptide is built upsequentially starting from the carboxy terminus and working toward theamino terminus of the peptide. Solid phase synthesis is begun bycovalently attaching the C terminus of a protected amino acid to asuitable resin, such as benzhydrylamine resin (BHA),methylbenzhydrylamine resin (MBHA) or chloromethyl resin (CMR), as isgenerally set forth in U.S. Pat. No. 4,244,946 or phenyl acid aminomethyl resin (PAM). A BHA or MBHA support resin is used if the carboxyterminus of the product peptide is to be a carboxamide. ACMR or PAMresin is generally used if the carboxy terminus of the product peptideis to be a carboxy group, although this may also be used to produce acarboxamide or ester.

The protective group on the a-amino group is removed by mild acidtreatment (i.e. trifluoroacetic acid). Suitable deprotection,neutralization and coupling cycles known in the art are used tosequentially add amino acids without isolation of the intermediate,until the desired peptide has been formed. The completed peptide maythen be deblocked and/or split from the carrying resin in any order.

Treatment of a resin supported peptide with HF or HBr/acetic acid splitsthe peptide from the resin and produces the carboxy terminal amino acidas a carboxylic acid or carboxamide.

If an ester is desired, the CMR or Pam resin may be treated with anappropriate alcohol, such as methyl, ethyl, propyl, butyl or benzylalcohol, in the presence of triethylamine to cleave the peptide from theresin and product the ester directly.

Esters of the peptides of this invention may also be prepared byconventional methods from the carboxylic acid precursor. Typically, thecarboxylic acid is treated with an alcohol in the presence of an acidcatalyst. Alternatively, the carboxylic acid may be converted to anactivated acyl intermediate, such as an acid halide, and treated with analcohol, preferably in the presence of a base.

The preferred method for cleaving a peptide from the support resin is totreat the resin supported peptide with anhydrous HF in the presence of asuitable cation scavenger, such as anisole or dimethoxybenzene. Thismethod simultaneously removes all protecting groups, except a thioalkylgroup protecting sulfur, and splits the peptide from the resin. Peptideshydrolyzed in this way from the CMR and Pam resins are carboxylic acids,those split from the BHA resin are obtained as carboxamides.

Modification of the terminal amino group of the peptide is accomplishedby alkylation or acylation by methods generally known in the art. Thesemodifications may be carried out upon the amino acid prior toincorporation into the peptide, or upon the peptide after it has beensynthesized and the terminal amino group liberated, but before theprotecting groups have been removed.

Typically, acylation is carried out upon the free amino group using theacyl halide, anhydride or activated ester, of the corresponding alkyl oraryl acid, in the presence of a tertiary amine. Mono-alkylation iscarried out most conveniently by reductive alkylation of the amino groupwith an appropriate aliphatic aldehyde or ketone in the presence of amild reducing agent, such a lithium or sodium cyanoborohydride.Dialkylation may be carried out by treating the amino group with anexcess of an alkyl halide in the presence of a base.

Solution synthesis of peptides is accomplished using conventionalmethods used to for amide bonds. Typically, a protected t-Boc amino acidwhich has a free carboxyl group is coupled to a protected amino acidwhich has a free amino group using a suitable coupling agent, such asN,N'-dicyclohexyl carbodiimide (DCC), optionally in the presence ofcatalysts such as 1-hydroxybenzotriazole (HOBT) or dimethylaminopyridine (DMAP). Other methods, such as the formation of activatedesters, anhydrides or acid halides, of the free carboxyl of a protectedt-Boc-amino-acid, and subsequent reaction with the free amine of aprotected amino acid, optionally in the presence of a base, are alsosuitable. For example, a protected Boc-amino acid or peptide is treatedin an anhydrous solvent, such as methylene chloride or tetrahydrofuran(THF), in the presence of a base, such as N-methyl morpholine, DMAP(dimethylaminopyridine) or a trialkyl amine, with isobutyl chloroformateto form the "activated anhydride", which is subsequently reacted withthe free amine of another protected amino acid or peptide. The peptideformed by these methods may be deprotected selectively, usingconventional techniques, at the amino or carboxy terminus and coupled toother peptides or amino acids using similar techniques. After thepeptide has been completed, the protecting groups may be removed ashereinbefore described, such as by hydrogenation in the presence of apalladium or platinum catalyst, treatment with sodium in liquid ammonia,hydrofluoric acid or alkali.

If the final peptide, after it has been deprotected, contains a basicgroup, an acid addition salt may be prepared. Acid addition salts of thepeptides are prepared in a standard manner in a suitable solvent fromthe parent compound and a slight excess of an acid, such a hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic ormethanesulfonic. The acetate salt for is especially useful. If the finalpeptide contains an acidic group, cationic salts may be prepared.Typically the parent compound is treated with a slight excess of analkaline reagent, such as a hydroxide, carbonate or alkoxide, containingthe appropriate cation. Cations such as Na⁺, K⁺, Ca⁺⁺ and NH₄ ⁺ areexamples of cations present in pharmaceutically acceptable salts. Na⁺and NH₄ ⁺ are especially preferred.

In general, in order to exert a inhibitory effect, the peptides of theinvention may be administered to human patients by injection in the doserange of about 0.5 ng to about 10 mg, for example about 5-500 ng, ororally in the dose range of about 50 ng to about 5 mg, for example about0.1 ng to 1 mg per 70 kg body weight per day; if administered byinfusion or similar techniques, the dose may be in the range of about0.005 ng to about 10 mg per 70 kg body weight, for example about 0.03 ngto 1 mg over six days. In principle, it is desirable to produce aconcentration of the peptide of about 10⁻¹⁵ M to about 10⁻⁵ M in theextracellular fluid of the patient.

According to a still further feature of the present invention there areprovided pharmaceutical compositions comprising as active ingredient oneor more compound of formula (I) as hereinbefore defined orphysiologically compatible salts thereof, in association with apharmaceutical carrier or excipient. The compositions according to theinvention may be presented for example, in a form suitable for oral,nasal, parenteral or rectal administration.

As used herein, the term "pharmaceutical" includes veterinaryapplications of the invention. These peptides may be encapsulated,tableted or prepared in an emulsion or syrup for oral administration.Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Liquid carriers include syrup, peanut oil, olive oil,glycerin, saline and water. Solid carriers include starch, lactose,calcium sulfate dihydrate, terra alba, magnesium stearate or stearicacid, talc, pectin, acacia, agar or gelatin. The carrier may alsoinclude a sustained release material such a glyceryl monostearate orglyceryl distearate, alone or with a wax. The amount of solid carriervaries but, preferably will be between about 20 mg to about 1 g perdosage unit. The pharmaceutical preparations are made following theconventional techniques of pharmacy involving milling, mixing,granulating, and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. Capsulescontaining one or several active ingredients may be produced, forexample, by mixing the active ingredients with inert carriers, such aslactose or sorbitol, and filling the mixture into gelatin capsules. Whena liquid carrier is used, the preparation will be in the form of asyrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such aliquid formulation may be administered directly p.o. or filled into asoft gelatin capsule. Organ specific carrier systems may also be used.

Alternately pharmaceutical compositions of the peptides of thisinvention, or derivatives thereof, may be formulated as solutions oflyophilized powders for parenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqueous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or buffered sodium or ammonium acetate solution. Suchformulation is especially suitable for parenteral administration, butmay also be used for oral administration and contained in a metered doseinhaler or nebulizer for insufflation. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium chloride or sodiumcitrate.

For rectal administration, a pulverized powder of the peptides of thisinvention may be combined with excipients such as cocoa butter,glycerin, gelatin or polyethylene glycols and molded into a suppository.The pulverized powders may also be compounded with an oily preparation,gel, cream or emulsion, buffered or unbuffered, and administered througha transdermal patch.

Nasal sprays may be formulated similarly in aqueous solution and packedinto spray containers either with an aerosol propellant or provided withmeans for manual compression.

Dosage units containing the compounds of this invention preferablycontain 1 mg-100 mg, for example 0.1-50 mg of the peptide of formula (I)or salt thereof.

According to a still further feature of the present invention there isprovided a method of inhibition of myelopoiesis which comprisesadministering an effective amount of a pharmaceutical composition ashereinbefore defined to a subject.

No unacceptable toxicological effects are expected when compounds of theinvention are administered in accordance with the present invention.

Biological Assays

A. In Vitro Hematopoietic Assay

The following in vitro assay system quantitates positive or negativeeffects of the compounds of this invention on the entrance ofgranulocyte-macrophage progenitor cells (CFU-GM) into cell cyclefollowing pulse exposure in vitro. Under soft agar culture conditions inthe presence of appropriate hematopoietic growth factors, colonies ofgranulocytes and macrophages can be quantitated and arise specificallyfrom the proliferation of single CFU-GM. In order to proliferate, CFU-GMmust enter S-phase of the cell cycle. Exposure of bone marrow cells to ³H-Thymidine prior to the soft agar culture eliminates those CFU-GM whichare actively proliferating, ie, are in S-phase of the cell cycle. Thiseffect is quantitated by a decrease in total CFU-GM colonies quantitatedin the agar assay. In comparison to controls, the degree of decrease orincrease in colony counts provides an index of stimulation orinhibition, respectively, of CFU-GM proliferation.

Procedure:

Femoral bone marrow cells are obtained and suspended at a concentrationof 6-9×10⁶ cells/ml in McCoy's 5A medium supplemented with 10% fetalbovine serum, antibiotics, essential and non-essential amino acids, MEMvitamins, serine, asparagine, glutamine, sodium pyruvate and sodiumbicarbonate. Replicate cultures are pulsed for 2 hours with compounds ofthe invention over a concentration range of 1 pg to 1 mg per ml finalconcentration and incubated at 37° C. in an atmosphere of 7% CO₂ inhumidified air. Following incubation, the cultures are washed threetimes with assay medium to remove peptides, and exposed to 560 uCi of ³H-Thymidine (Specific Activity=20 Ci/mMole) for 20 minutes. Afterexposure, 0.5 ml of cold Thymidine (1 mg/ml stock solution) is added toeach tube to stop the reaction. Cultures are washed three times andplated in the CFU-GM agar assay at a concentration of 75,000 cells perculture in media as described above and containing 0.3% agar and 10% v/vmedium conditioned by spleen cells exposed to pokeweed mitogen as asource of hematopoietic growth factors. The cultures are incubated at37° C., 7% CO₂ in humidified air for 5-7 days and CFU-GM coloniesenumerated microscopically. The percentage of CFU-GM in S-phase iscalculated by dividing colony counts from ³ H-Thymidine treated culturesby those of untreated control cultures. These values are compared tosimilarly derived numbers from peptide exposed and untreated cells.

B. Measurement of Superoxide Formation

Polymorphonuclear neutrophils (PMNs) are the first-line defense againstmany invading pathogens including C. albicans. The assessment of ex vivocandidacidal-activity of PMNs from treated animals is important indetermining the mode of action of therapeutic compounds in the C.albicans animal model. Superoxide is the major initial product of oxygenreduction in the respiratory burst of activated PMNs and the variousspecies of oxygen radicals generated during the respiratory burst aredirectly or indirectly cytotoxic to invading pathogens. Therefore, themeasurement of superoxide formation and release during stimulation isimportant in the assessment of the oxidative metabolism and subsequentcidal efficiency of phagocytic cells.

Procedure:

The compound or control is administered to normal mice IP daily for 7days. Peripheral blood from individual treated animals was collected andpooled. PB-PMNs were purified by standard separation methods; (Boyum, A.1968. "Isolation of mononuclear cells and granulocytes from humanperipheral blood" Scand. J. Clin. Lab Invest. 21: 77-89); and cellsuspensions from the different groups were standardized to containequivalent numbers of PMNs/ml. For candidacidal assays sufficientPB-PMNs were added to quadruplicate tubes containing C. albicans yeastsand 10% normal mouse serum in balanced salt solution to result in afinal E:T ratio of 10:1. PB-PMNs were omitted from control tubes. Thetubes were incubated at 37° C. for 1 hr. The phagocytes were lysed andaliquots were plated onto agar plates. The plates were incubated for 48hrs at which time the number of colony forming units (CFU) wereenumerated and the percent reduction in CFU for each sample determined.

Superoxide dismutase-inhibitable superoxide anion production wasquantitated in a microtiter ferricytochrome c reduction assay (1.5×10⁵PB-PMN/well) in 0.5% gelatin/balanced salt solution during exposure tosoluble and particulate stimuli (phorbol 12-myristate 13-acetate (PMA)and serum-opsonized C. albicans, respectively). The nmoles offerricytochrome c reduced/well (quadruplicate wells/pooled group) wasdetermined following a 1 hr incubation. Baseline superoxide activity wasdetermined in the absence of any stimuli. PMA and Candida stimulatedactivity were corrected for spontaneous superoxide production prior todata calculation. Data for PB-PMNs from each treatment group areexpressed as the percent of control superoxide where control activity isthat observed for PB-PMNs from PBS/serum-treated mice.

The examples which follow serve to illustrate this invention. TheExamples are intended to in no way limit the scope of this invention,but are provided to show how to make and use the compounds of thisinvention.

In the examples, all temperatures are in degrees Centigrade. Amino acidanalysis were performed upon a Dionex Autoion 100. Analysis for peptidecontent is based upon Amino Acid Analysis. FAB mass spectra wereperformed upon a VG ZAB mass spectrometer using fast atom bombardment.The abbreviations used are as follows:

Abu=Aminobutyric acid

Ala=alanine

Asp=aspartic acid

t-Boc=tert. butyloxy carbonyl

Cad=cadaverine

Cl-Z=p-chlorobenzyloxy carbonyl (Z=benzyloxy carbonyl)

DCC=dicyclohexyl carbodiimide

DCM=dichloromethane

DIEA=diisopropylethyl amine

EDC=(N-ethyl-N'-)3-dimethylaminopropyl) carbodimide

Glu=glutamic-acid

p-Glu=pyroglutamic acid

Tyr=tyrosine

Hna=diaminohexynoic acid

HOBT=hydroxybenzotriazole

Lys=lysine

NMP=N-methyl-2-pyrrolidinone

Pic=picolenic acid

Pro=proline

Ppg=propargyl glycine

Gln=glutamine

Gly=glycine

Orn=ornithine

EXAMPLE 1

(pGlu-Glu-Asp-Abu-Lys): (SEQ ID NO. 1) Mol. Form C₂₄ H₃₈ N₆ O₁₁ Mol. Wt.585.6

800 mg of BOC-Lys (Cl-Z)-PAM resin (0.5 mM, substitution=0.63 mM/g) wascharged in an ABI Model 430 A Peptide Synthesizer. The target peptidewas synthesized according to manufacturer suggested standard protocolsusing 2 mM of each amino acid. (Applied Biosystems Model 430A PeptideSynthesizer Users Manual Version 1-30, February 1987, Part number000066). After completion of the synthesis, the resin peptide was washedwith DCM and dried (1.665 g). The peptide resin (1.2 g) was charged in acleavage apparatus and cleaved using 10 ml HF and 1 ml anisole at -15°C. for two hrs. After removal of HF, the resin was extensively washedwith ether and the peptide extracted in glacial acetic acid. Most of theacetic acid was removed on a rotovap and the residue was diluted withwater and lyophilized. Two hundred twenty milligrams of crude peptidewere obtained. The pure peptide (130 mg) was obtained after columnchromatography on a C-18 "Bond Elute"® column usingacetonitrile/water/trifluoroacetic acid (TFA) buffer. Amino acidanalysis: Asp 1.0 (1), Glu 1.98 (2), Abu 1.12(1), Lys 1.06 (1) FAB/MSMH⁺ 587.5

EXAMPLE 2

(Pic-Glu-Asp-Abu-Lys) (SEQ ID NO: 2) Mol. Form C₂₅ H₃₆ N₆ O₁₀ Mol. Wt580.6:

800 mg of BOC-Lys (Cl-Z)-PAM resin (0.5 mM/substitution 0.63 mM/g) wascharged in an ABI Model 430 A Peptide Synthesizer. The target peptidewas synthesized according to manufacturer suggested standard protocolsusing 2 mm of each amino acid. After completion of the synthesis, theresin peptide was washed with DCM and dried (1.6 g). The peptide resin(884 mg) was charged in the HF cleavage apparatus and cleaved using 10ml HF and 1 ml anisole at -15° C. for two hrs. After removal of HF, theresin was extensively washed with ether and the peptide was extracted inglacial acetic acid. Most of the acetic acid was removed on a rotavapand the residue was diluted with water and lyophilized. Two hundred andfifty-one milligrams of crude peptide were obtained. After purificationon a C-18 "Bond Elute"® column using acetonitrile/water/TFA buffer, 141mg of pure peptide (HPLC purity 97%) was obtained. Amino acid analysis:Asp 1.0 (1), Glu 1.03 (1), Abu 1.12 (1), Lys 1.17 (1). Pic is notidentified by amino acid analysis. FAB/MS 581.1

EXAMPLE 3

(Pic-Ser-Asp-Abu-Lys) (SEQ ID NO: 3) Mol. Form. C₂₃ H₃₄ N₆ O₉ Mol. Wt538.56:

Eight hundred fifty mg BOC-Lys (Cl-Z)-PAM resin (0.56 mm of resin) wascharged in an ABI Model 430 A Peptide Synthesizer. The target peptidewas synthesized according to manufacturer suggested standard protocolsusing 2 mm of each amino acid. After completion of the synthesis, theresin peptide was washed with DCM and dried (1.038 g). The peptide resin(1.03 g) was charged in the HF cleavage apparatus and cleaved using 10ml HF and 1 ml anisole at -15° C. for two hrs. After removal of HF, theresin was extensively washed with ether and the peptide was extracted intrifluoroacetic acid. Most of the trifluoroacetic acid was removed on arotavap and the residue was diluted with water and lyophilized. Twohundred and ninety-eight milligrams of crude peptide was obtained. Afterpurification on a C-18 "Bond Elute"® column using acetonitrile/water/TFAbuffer, 178 mg of pure peptide (HPLC purity 97%) was obtained. Aminoacid analysis: Asp 1.0 (1), Ser 0.88 (1), Abu 1.12 (1), Lys 1.17 (1).Pic is not identified by amino acid analysis.

EXAMPLE 4

(pGlu-Glu-Asp-Abu-Tyr-Lys): (SEQ ID NO: 4) Mol. Form. C₃₃ H₄₇ N₇ O₁₃Mol. Wt 749.77

Eight hundred fifty mg BOC-Lys (Cl-Z)-PAM resin (0.56 mm of resin) wascharged in ABI Model 430 A Peptide Synthesizer. The target peptide wassynthesized according to the manufacturer's suggested standard protocolsusing 2 mm of each amino acid. After completion of the synthesis, theresin peptide was washed with DCM and dried (1.353 g). The peptide resin(1.3 g) was charged in the HF cleavage apparatus and cleaved using 10 mlHF and 1 ml anisole at -15° C. for two hrs. After removal of HF, theresin was extensively washed with ether and the peptide was extracted intrifluoroacetic-acid. Most of the trifluoroacetic acid was removed on arotavap and the residue was diluted with water and lyophilized. Fourhundred and fifty-four milligrams of crude peptide were obtained. Thecrude peptide (204.7 mg) was purified on a C-18 "Bond Elute"® columnusing acetonitrile/water/TFA buffer. 95.2 mg of peptide (HPLC purity90%) was obtained. Amino acid analysis: pGlu+Glu 2.02 (2), Asp 1.0 (1),Tyr 0.94 (1), Lys 1.03 (1)

EXAMPLES 5-11

By the methods given above the following peptides were made.

Example

5 p-Glu-Glu-Asp-Cys-Gly-OH (SEQ ID NO: 5)

6 pGlu-Glu-Asp-Ser-Gly (SEQ ID NO: 6)

7 Pic-Glu-Asp-Abu-Lys-OH (SEQ ID NO: 7)

8 Pic-Ser-Asp-Abu-Lys-OH (SEQ ID NO: 8)

9 pGlu-Glu-Asp-Abu-Lys-OH (SEQ ID NO: 9)

10 pGlu-Glu-Asp-Abu-Tyr-Lys-OH (SEQ ID NO: 10)

11 pGlu-Glu-Asp-Abu-Lys-Tyr-OH (SEQ ID NO: 11)

EXAMPLE 12

Formulations for pharmaceutical use incorporating compounds of thepresent invention can be prepared in various forms and with numerousexcipients. Examples of such formulations are given below.

Tablets/Ingredients

Per Tablet

    ______________________________________                                        1.        Active ingredient                                                                              40    mg                                                     (Cpd of Form. I)                                                    2.        Corn Starch      20    mg                                           3.        Alginic acid     20    mg                                           4.        Sodium alginate  20    mg                                           5.        Mg stearate      1.3   mg                                                                      2.3   mg                                           ______________________________________                                    

Procedure for tablets:

Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitablemixer/blender.

Step 2 Add sufficient water portion-wise to the blend from Step 1 withcareful mixing after each addition. Such additions of water and mixinguntil the mass is of a consistency to permit its converion to wetgranules.

Step 3 The wet mass is converted to granules by passing it through anoscillating granulator using a No. 8 mesh (2.38 mm) screen.

Step 4 The wet granules are then dried in an oven at 140° F. (60° C.)until dry.

Step 5 The dry granules are lubricated with ingredient No. 5.

Step 6 The lubricated granules are compressed on a suitable tabletpress.

Parenteral Formulation

A pharmaceutical composition for parenteral administration is preparedby dissolving an appropriate amount of a compound of formula I inpolyethylene glycol with heating. This solution is then diluted withwater for injections Ph Eur. (to 100 ml). The solution is thensterilized by filtration through a 0.22 micron membrane filter andsealed in sterile containers.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1 and 4                                                         (D) OTHER INFORMATION: /note= "First Xaa = pyroglutanic                       acid, second is aminobutyric acid"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       XaaGluAspXaaLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1 and 4                                                         (D) OTHER INFORMATION: /note= "First Xaa is picolinic                         acid, second is aminobutyric acid"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaGluAspXaaLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1 and 4                                                         (D) OTHER INFORMATION: /note= "First Xaa is picolinic                         acid, second is aminobutyric acid"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       XaaSerAspXaaLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1 and 4                                                         (D) OTHER INFORMATION: /note= "First Xaa is pyroglutanic                      acid, second is aminobutyric acid"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       XaaGluAspXaaTyrLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1 and 5                                                         (D) OTHER INFORMATION: /note= "First Xaa is pyroglutamic                      acid, second is Gly-OH"                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       XaaGluAspLysXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "Xaa is pyrolutanic acid"                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       XaaGluAspSerGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1 and 5                                                         (D) OTHER INFORMATION: /note= "First Xaa is picolinic                         acid, second is Lys-OH"                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       XaaGluAspSerXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1, 4 and 5                                                      (D) OTHER INFORMATION: /note= "First Xaa is picolinic                         acid, second is aminobutyric acid, third is Lys-OH"                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       XaaSerAspXaaXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1, 4 and 5                                                      (D) OTHER INFORMATION: /note= "First Xaa is pyroglutamic                      acid, second is aminobutyric acid, third is Lys-OH"                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       XaaGluAspXaaXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1, 4 and 6                                                      (D) OTHER INFORMATION: /note= "First Xaa is pyrogultamic                      acid, second is aminobutyric acid, third is Lys-OH"                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      XaaGluAspXaaTyrXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1, 4 and 6                                                      (D) OTHER INFORMATION: /note= "First Xaa is pyroglutamic                      acid, second is aminobutyric acid, third is Tyr-OH"                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      XaaGluAspXaaLysXaa                                                            15                                                                            __________________________________________________________________________

We claim:
 1. A compound of formula I:

    A-B-C-D-(X).sub.n -E                                       (I)

wherein A is picolinic acid, pyroglutamic acid, proline or L-pipecolinicacid; B is serine, glutamic acid, aspartic acid, threonine, cysteine ortyrosine; C is aspartic acid or glutamic acid; D is aminobutyric acid,alanine, glycine, or serine; X is tyrosine or lysine; n is 0 or 1; E is--N(R₁)--R₂ R₃ or E is lysine, ornithine, tyrosine,para-aminophenylalanine or the carboxamide or hydroxymethyl derivativethereof; R₁, R₄ and R₅ are independently hydrogen, C₁₋₅ alkyl, phenyl,napthyl or C₅₋₇ cycloalkyl; R₂ is C₁₋₆ alkyl, phenyl, napthyl or C₅₋₇cycloalkyl; R₃ is --NR₄ R₅ or a pharmaceutically acceptable saltthereof.
 2. A compound of claim 1 wherein A is pyroglutamic acid orpicolinic acid; B is glutamic acid or serine; C is aspartic acid; D isaminobutyric acid; E is glycine or lysine and n is
 0. 3. A compound ofclaim 1 wherein the chiral amino acids are in the L absoluteconfiguration.
 4. A compound of claim 1 wherein n is 1 and E is glycineor lysine.
 5. A compound of claim 1 selected from the group consistingof:

    Pic-Glu-Asp-Abu-Lys (SEQ ID NO: 2)

    pGlu-Glu-Asp-Abu-Tyr-Lys (SEQ ID NO: 4)

    pGlu-Glu-Asp-Abu-Lys (SEQ ID NO: 1)

    Pic-Ser-Asp-Abu-Lys (SEQ ID NO: 3).


6. 6. A pharmaceutical composition comprising a compound of claim 1 anda pharmaceutical acceptable carrier.
 7. A method of inhibiting themyelopoietic system which comprises administering to a subject in needthereof, an effective amount of a compound of claim 1.